These techniques have been widely adopted by research groups involved in microbial cell biology, and the plasmid vectors have now been distributed directly to well over 100 laboratories world-wide, although they are now also distributed by the Bacillus Genetic Stock Centre. These techniques were instrumental in determining the establishment of compartment specificity of developmentally regulated s-factors during sporulation in B. subtilis (Lewis et al., PNAS 91, 3849-3853 (1994)). I also showed that prespore specific accumulation of the transcription regulator SpoIIAA was responsible for initiation of compartment-specific gene expression during development, that this accumulation was probably due to prespore-specific activity of the phosphatase SpoIIE, and that a programme of proteolysis was initiated following this activation event (Lewis et al., Genes Cell 1, 881-894 (1996); Lewis et al., J. Bacteriol 180, 3276-3284 (1998)). I was also involved in work that showed that the highly conserved SpoIIIE (FtsK) protein is a DNA translocase that moves DNA through a division septum (Wu et al., Genes Dev 9, 1316-1326 (1995)). This was the first example of such a phenomenon, and was a very significant finding as previously it was assumed that DNA was segregated into daughter cells/different compartments prior to division septum formation. Finally, I have shown that transcription and translation are spatially separated within bacteria (Lewis et al., EMBO J 19, 710-718 (2000)). This was an unexpected result as the 2 processes were thought to be very tightly coupled in bacteria. Furthermore, transcription becomes concentrated into a sub-fraction of the bacterial nucleoid at higher growth rates. These transcription foci have been shown to be the sites of rRNA synthesis, and my laboratory is now focussing on characterising transcription complexes and their structure in detail.

Research ExpertiseMy research career has focused on the gram positive bacterium Bacillus subtilis. During my PhD I purified and characterised the replication terminator protein (RTP) and its DNA binding sites (Lewis et al., J. Bacteriol 171, 3564-3567 (1989); Lewis et al., J. Mol. Biol. 214, 73-84 (1990)). These sites are regions of DNA where oppositely moving replication forks meet and newly replicated chromosomes are resolved prior to segregation. During my post-doctoral research I was responsible for the development of cell biological techniques and vectors for visualising gene expression and protein localisation in live bacterial cells (Lewis et al., PNAS 91, 3849-3853 (1994); Lewis et al., Mol Microbiol 13, 655-662 (1994); Lewis and Errington, Microbiology 142, 733-740 (1996); Lewis and Marston, Gene 227, 101-109 (1999); Feucht and Lewis, Gene 264, 289-297 (2001)). These techniques have been widely adopted by research groups involved in microbial cell biology, and the plasmid vectors have now been distributed directly to well over 100 laboratories world-wide, although they are now also distributed by the Bacillus Genetic Stock Centre. These techniques were instrumental in determining the establishment of compartment specificity of developmentally regulated s-factors during sporulation in B. subtilis (Lewis et al., PNAS 91, 3849-3853 (1994)). I also showed that prespore specific accumulation of the transcription regulator SpoIIAA was responsible for initiation of compartment-specific gene expression during development, that this accumulation was probably due to prespore-specific activity of the phosphatase SpoIIE, and that a programme of proteolysis was initiated following this activation event (Lewis et al., Genes Cell 1, 881-894 (1996); Lewis et al., J. Bacteriol 180, 3276-3284 (1998)). I was also involved in work that showed that the highly conserved SpoIIIE (FtsK) protein is a DNA translocase that moves DNA through a division septum (Wu et al., Genes Dev 9, 1316-1326 (1995)). This was the first example of such a phenomenon, and was a very significant finding as previously it was assumed that DNA was segregated into daughter cells/different compartments prior to division septum formation. Finally, I have shown that transcription and translation are spatially separated within bacteria (Lewis et al., EMBO J 19, 710-718 (2000)). This was an unexpected result as the 2 processes were thought to be very tightly coupled in bacteria. Furthermore, transcription becomes concentrated into a sub-fraction of the bacterial nucleoid at higher growth rates. These transcription foci have been shown to be the sites of rRNA synthesis, and my laboratory is now focussing on characterising transcription complexes and their structure in detail.

Invitations

Participant

ReviewerOrganisation: Various Publishers in the field of Environmental and Life Science
Description:
Have been invited to write reviews for top journals in field (Molecular Microbiology, Microbiology and international Review of Cytology) and 2 book chapters in the last 3 years. Work is also now appearing in Microbiology text books.

Teaching

Code

Course

Role

Duration

BIOL3090

Molecular BiologyFaculty of Science and IT, University of Newcastle

Course coordinator/lecturer

21/02/2002 - 21/08/2016

BTEC3250

Biotech PlacementFaculty of Science and IT, University of Newcastle

Course coordinator (2012)/supervisor

21/07/2001 - 21/08/2016

BIOL3001

Advanced Lab Skills in BiologyFaculty of Science and IT, University of Newcastle

Course coordinator/Lecturer

21/02/2007 - 21/08/2016

BIOL2002

Lab Skills in BiologyFaculty of Science and Information Technology, University of Newcastle

Lecturer

21/07/2008 - 31/12/2012

BIOL3100

MicrobiologyFaculty of Science and Information Technology, University of Newcastle

Course coordinator/lecturer

21/02/2016 - 21/08/2016

BIOL1003

Biology Professional Skills 1Faculty of Science and Information Technology, University of Newcastle

Lecturer

21/07/2011 - 21/08/2015

BIOL2090

Microbial BiologyFaculty of Science and Information Technology, University of Newcastle

Our ongoing research focused on targeting transcription initiation in bacteria has resulted in synthesis of several classes of mono-indole and mono-benzofuran inhibitors that targ... [more]

Our ongoing research focused on targeting transcription initiation in bacteria has resulted in synthesis of several classes of mono-indole and mono-benzofuran inhibitors that targeted the essential protein-protein interaction between RNA polymerase core and s 70/s A factors in bacteria. In this study, the reaction of indole-2-, indole-3-, indole-7- and benzofuran-2-glyoxyloyl chlorides with amines and hydrazines afforded a variety of glyoxyloylamides and glyoxyloylhydrazides. Similarly, condensation of 2- and 7-trichloroacetylindoles with amines and hydrazines delivered amides and hydrazides. The novel molecules were found to inhibit the RNA polymerase-s 70/s A interaction as measured by ELISA, and also inhibited the growth of both Gram-positive and Gram-negative bacteria in culture. Structure-activity relationship (SAR) studies of the mono-indole and mono-benzofuran inhibitors suggested that the hydrophilic-hydrophobic balance is an important determinant of biological activity.

We have examined the localization of DNA replication of the Bacillus subtilis phage f29 by immunofluorescence. To determine where phage replication was localized within infected c... [more]

We have examined the localization of DNA replication of the Bacillus subtilis phage f29 by immunofluorescence. To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA. On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane. Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid. Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7.

Using fusions of green fluorescent protein to subunits of RNA polymerase (RNAP) and ribosomes, we have investigated the subcellular localization of the transcriptional and transla... [more]

Using fusions of green fluorescent protein to subunits of RNA polymerase (RNAP) and ribosomes, we have investigated the subcellular localization of the transcriptional and translational machinery in the bacterium Bacillus subtilis. Unexpectedly, we found that RNAP resides principally within the nucleoid. Conversely, ribosomes localized almost exclusively outside the nucleoid, concentrating particularly towards sites of cell division. This zonal localization was not dependent on cell division and is probably due, at least in part, to exclusion from the nucleoid. Dual labelling of RNAP and ribosomes was used to confirm the spatial separation of the two processes. We conclude that, even in the absence of a nuclear membrane, transcription and translation occur predominantly in separate functional domains. At higher growth rates, concentrations of RNAP developed, probably representing the sites of rRNA synthesis. These may represent a further spatial specialization, possibly equivalent to the eukaryotic nucleolus.

We report the development of a series of plasmid vectors for the construction of fusions to mutants of the intrinsically fluorescent green fluorescent protein, GFPmut1 (Cormack et... [more]

We report the development of a series of plasmid vectors for the construction of fusions to mutants of the intrinsically fluorescent green fluorescent protein, GFPmut1 (Cormack et al., 1996. Gene 173, 33-38) and GFPuv (Crameri et al., 1996. Nature Biotechnology 14, 315-319). Both N- and C-terminal fusions can be produced, and their expression can be finely controlled from the inducible Pxyl promoter following double crossover integration into the amyE locus of the Bacillus subtilis chromosome. Other vectors designed for single crossover insertion into the chromosome allow downstream genes to be placed under inducible control. We also show that fusions to GFPmut1 and GFPuv can be co-localized within the cell by virtue of their different excitation spectra.

Immunofluorescence microscopy was used to study the establishment of compartment-specific transcription during sporulation in Bacillus subtilis. Analysis of the distribution of th... [more]

Immunofluorescence microscopy was used to study the establishment of compartment-specific transcription during sporulation in Bacillus subtilis. Analysis of the distribution of the anti-anti-sigma factor, SpoIIAA, in a variety of mutant backgrounds supports a model in which the SpoIIE phosphatase, which activates SpoIIAA by dephosphorylation, is sequestered onto the prespore face of the asymmetric septum. Thus, prespore-specific gene expression apparently arises as a result of the compartmentalization of SpoIIE protein. The results also suggest the existence of at least two compartment-specific programs of proteolysis, one dependent on the mother cell-specific sigma factor s(E) and the other dependent on the prespore- specific sigma factor s(F).

Spore formation in Bacillus subtilis begins with an asymmetric cell division that superficially resembles the division of vegetative cells. Mutations in the spoIIIE gene of B. sub... [more]

Spore formation in Bacillus subtilis begins with an asymmetric cell division that superficially resembles the division of vegetative cells. Mutations in the spoIIIE gene of B. subtilis partially block partitioning of one chromosome into the smaller (prespore) compartment of the sporulating cell. Point mutations that specifically block prespore chromosome partitioning affect a carboxy-terminal domain of SpoIIIE that shows significant sequence similarity to the DNA transfer (Tra) proteins of several conjugative plasmids of Streptomyces. In wild-type sporulating cells, the prespore chromosome passes through an intermediate stage resembling the state in which spoIIIE mutant cells are blocked. The prespore chromosome is then transferred progressively through the newly formed spore septum. We propose that translocation of the prespore chromosome occurs by a mechanism that is functionally related to the conjugative transfer of plasmid DNA.

Lewis PJ, Partridge SR, Errington J, 's Factors, asymmetry, and the determination of cell fate in Bacillus subtilis', Proceedings of the National Academy of Sciences of the United States of America, 91 3849-3853 (1994)

Soon after the initiation of sporulation, Bacillus subtilis divides asymmetrically to produce sister cells that have very different developmental fates. Recently, it has been prop... [more]

Soon after the initiation of sporulation, Bacillus subtilis divides asymmetrically to produce sister cells that have very different developmental fates. Recently, it has been proposed that the differential gene expression ywhich begins soon after this division is due to cell-specific activation of the transcription factors s(F) and s(E) in the prespore and the mother cell, respectively. We describe the use of a method for the localization of gene expression in individual sporulating cells that lends strong support to the cell-specific localization of s(F) and s(E) activities. The dependence of s(E) activity on integrity of the gene encoding s(F) has led to the suggestion that activation of s(F) in the prespore leads to a directional signal that triggers activation of s(E) only in the mother cell. Here we show that s(E) actually specifies the fate of the mother cell; in the absence of s(E), two prespore-like cells are made. The appearance of s(F) activity at both poles of a s(E)-deficient mutant supports the idea that s(F) normally remains latent in the mother cell and that its activation depends on some morphological or physiological feature of the prespore. We present a model for the generation of asymmetry and the establishment of cell fate in B. subtilis.

Citations

Scopus - 70

1994

Lewis PJ, Partridge SR, Errington J, 'Sigma factors, asymmetry and the determination of cell fate in Bacillus subtilis', Proceedings of the National Academy of Sciences of USA, 91 3849-3853 (1994)

Transcription initiation/recycling 2007 - 2016

Transcription termination complexes 2015 - 2016

Research Collaborations

The map is a representation of a researchers co-authorship with collaborators across the globe. The map displays the number of publications against a country, where there is at least one co-author based in that country. Data is sourced from the University of Newcastle research publication management system (NURO) and may not fully represent the authors complete body of work.